DESCRIPTION: The overall goal of the project described in this proposal is the determination of the structure function relationships of the heme binding site in the protein HbpA from Haemophilus influenza. H. influenza strains have an absolute growth requirement for heme and yet the mechanism by which this pathogen obtains its heme from its host is not understood. Heme binding protein A (HbpA) from Haemophilus influenza appears to play a role in the mechanism of heme scavenging and/or transport in this pathogen and hence could be an important pharmacological target. Dr. Lukat-Rodgers proposes to utilize UV-visible, ESR, and resonance Raman spectroscopies to determine the oxidation state(s) and coordination number of the heme iron, the identity of the heme axial ligand(s), and hydrophobicity of the heme pocket in HbpA. The affinity of HbpA for heme will be measured by titration experiments and the kinetics of the heme binding reaction of HbpA will be examined. These thermodynamic and kinetic parameters, coupled with analysis of the heme environment in heme:HbpA complex, are suggested to provide significant insight into the mechanism of Haemophilus influenza heme acquisition. Specific aims of the proposal include: 1) Spectroscopic characterization of the heme and its environment in heme:HbpA. UV-visible, ESR, and resonance RR spectroscopies will be employed to elucidate structural features of the heme and the heme pocket. Specifically, the questions of the oxidation state(s) and coordination number of the heme iron, the identity of the heme axial ligand(s), and hydrophobicity of the heme pocket will be addressed with these techniques. 2) Thermodynamic and kinetic analyses of heme binding by HbpA will be ascertained through titrations of apoHbpA with hemin. The spectroscopic features (UV-visible and resonance Raman signatures) determined in the first specific aim will be used to monitor the reaction kinetics of heme binding to HbpA. Mechanistic details will be obtained via examination of the effect of heme iron oxidation state, heme iron axial ligation, temperature, and pH on the rate of heme binding to HbpA.